Czech Society for Mass Spectrometry

(CEEPC/IPM/CMSC 2022 - ThS-02)
Optimized HDX-MS workflow for antibody structure monitoring by HDX-MS

Zuzana Kalaninová ^1,2 *, Barbora Jirečková ^1,2, Lukáš Fojtík ^1,2, Michael Volný ¹, Josef Chmelík ¹, Petr Novák ¹, Petr Man ¹

1. BioCeV - Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic
2. Faculty of Science, Charles University, Prague, Czech Republic

Abstract

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is one of the most prominent and versatile techniques providing unique structural information. The main advantages of this method are no limitations in protein-size and compatibility with any experimental conditions such as pH, temperature, protein concentration, or buffer composition. HDX-MS is also officially approved by legal authorities as a suitable tool for validation of protein-based biotherapeutic molecules, including monoclonal antibodies. However, antibodies are challenging analytical targets due to their N-glycosylation and compact structure stabilized by disulfide bonds.

In this study, we systematically evaluated crucial steps in the initial phase of the HDX experiment. This included testing of various quench conditions - disulphide bond reduction and subsequent proteolysis where different proteases, alone or in combination were utilized. We also developed detailed data processing workflow that helps in complete understanding of digest parametrization and easy cross-comparison. The final, fully optimized conditions provided full sequence coverage, optimal peptide length and reasonable redundancy and thus improved spatial resolution.

* Corresponding author: kalaninova.z@gmail.com

Acknowledgement:

Financial support from CSF project 22-27695S and CIISB (LM2018127) is gratefully acknowledged.